Hi,
I hope someone can answer my questions and bring some light into my darkness.
I am using the NEXTflex Small RNA Kit in CLIP experiments to analyze mRNA binding motifs. So I immunoprecipitate RNP, do a proteinase K treatment, and precipitate previously bound RNA by TRI reagent. It work well so far and after using that Kit I got amplified products between 140 and 180 nts.
So far, so good.
Yesterday I though I might need a positiv and a negative control, respectively ... just checking! So I used the microRNA control (part of the kit) as positive control and added just water as negativ control (for ligations steps). After the second ligation I gel-purified my products by using a 12% UreaPA gel ... mainly to get rid of the adapterdimer. Unfortunately, I obtain a "postive band" in both samples after PCR, means for the 21 nt microRNA and the negative control "water".
How can that be!? My problem is that I already got some products for sequencing, but now I am not sure if I have real data or just this "postive negative control" thing. Is someone able to advice me somehow!? That would be endless generous
Thanks a lot.
Best, Carlo
I hope someone can answer my questions and bring some light into my darkness.
I am using the NEXTflex Small RNA Kit in CLIP experiments to analyze mRNA binding motifs. So I immunoprecipitate RNP, do a proteinase K treatment, and precipitate previously bound RNA by TRI reagent. It work well so far and after using that Kit I got amplified products between 140 and 180 nts.
So far, so good.
Yesterday I though I might need a positiv and a negative control, respectively ... just checking! So I used the microRNA control (part of the kit) as positive control and added just water as negativ control (for ligations steps). After the second ligation I gel-purified my products by using a 12% UreaPA gel ... mainly to get rid of the adapterdimer. Unfortunately, I obtain a "postive band" in both samples after PCR, means for the 21 nt microRNA and the negative control "water".
How can that be!? My problem is that I already got some products for sequencing, but now I am not sure if I have real data or just this "postive negative control" thing. Is someone able to advice me somehow!? That would be endless generous
Thanks a lot.
Best, Carlo
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